Anti-inflammatory peptides

ABSTRACT

Methods for inhibiting an inflammatory reaction in a mammal and pharmaceutical compositions are provided. The methods comprise administering to the mammal an effective amount of a peptide of the formula:  
     X 1 —X 2 —X 3  wherein X 1  is selected from the group consisting of 2-amino-hexanoic acid; 2-amino-heptanoic acid; 2-amino-octanoic acid; cyclohexyl-substituted 2-amino-ethanoic acid, 2-amino-propanoic acid or 2-amino-butanoic acid and methionine; X 2  is an acidic amino acid; and X 3  is an aliphatic amino acid; or of a peptide of the formula: X 4 —X 5  wherein X 4  is an aromatic or aliphatic amino acid; and X 5  is an acidic amino acid.

RELATED APPLICATION INFORMATION

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/353,231, filed Feb. 4, 2002; the disclosure of whichis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to dipeptides and tripeptides withanti-inflammatory activity and to methods and compositions for treatinginflammatory reactions, including allergic reactions.

BACKGROUND OF THE INVENTION

[0003] Immediate or Type 1 allergic reactions generally occur, insusceptible humans and animals, immediately following contact with anantigen, which is referred to as the allergen. Such reactions may havelong term consequences. Type I allergic reactions are largely attributedto IgE antibodies, although IgG antibodies can participate in andmodulate them (Dombrowicz et al, 1998; Ujike et al, 1999). An allergicreaction is generally caused by the activation of a subpopulation ofimmune cells, the mast cells and basophils. When an antigen reacts withIgE antibody receptors on the surface of a cell, chemical mediatorsinitiate the allergic reaction by acting on adjacent immune, epithelial,endothelial and smooth muscle cells and promote, in a longer term, theinflux of other inflammatory and immune cells (neutrophils, eosinophils,monocytes, lymphocytes) into the affected tissue. This influx ofinflammatory cells predisposes the patient to recurrent, and sometimesdelayed or prolonged, allergic or hypersensitivity reactions and isfrequently associated with non-reversible remodelling of the tissue.Type 1 allergic reactions are identified according to the location inwhich they occur, e.g. asthmatic reactions occur in the lungs, rhinitisin the nose, conjunctivitis in the eyes, atopic dermatitis in the skin,systemic allergic reactions in the circulation and intestinal reactionsin the gastrointestinal system.

[0004] Asthma can be defined clinically as a condition of intermittent,reversible airways obstruction; asthma manifests itself as severalclinical entities: allergic asthma, bronchial asthma, exercise inducedasthma (EIA), chemical induced asthma, and status asthmaticus. Asthmacan be divided into two types. Extrinsic asthma is generally triggeredby external agent such as allergens (dust mites, pollen, stings, drugs,or foods), and is commonly diagnosed in early life. Intrinsic asthma,which is allergic-like and generally develops later in life, can betriggered by congested and inflamed tissues, infection, common coldviruses (RSV, parainfluenza, rhinovirus), endogenous catecholamines(e.g. adrenaline), drugs (e.g. aspirin), cold air, stress or exertion,various irritants (cigarette smoke, burning leaves, perfumes, strongodors) and some food intolerance. Intrinsic asthma occurs without theparticipation of an allergic trigger by specific antibodies against anallergen.

[0005] Bronchial asthma is usually a chronic (long-term) diseaseaffecting the bronchial tubes (bronchi and/or airways) of the lungs. Thesymptoms of asthma are the result of constriction or narrowing ofirritable bronchial tubes resulting in wheezing and difficulty inbreathing. This constriction of the airways, caused by bronchial tubemuscle spasm and narrowing due to inflammation, is most frequentlyprovoked by antigen-induced release of histamine, leukotrienes and otherchemical mediators from mast cells. With intrinsic asthma, thesemediators are released without an allergic trigger. The net result ofthe asthmatic attack is muscle spasm, inflammation, edema (swelling),and increased mucus production within the bronchi. Both extrinsic andintrinsic asthmatic reactions generate symptoms within 15-30 minutes ofexposure (immediate response) which generally subside within an hour. Insome individuals, a delayed response (late phase reaction) occurs 3-4hours following the immediate or initial response. It should be noted,however, that the timing of these reactions exhibits marked variabilitybetween patients and can be shorter or longer in onset and duration. Thelate phase reaction, which probably develops from an inflammatoryreaction, may last many hours or days and is frequently associated withincreased bronchial hyperreactivity or persistent airwayhyperresponsiveness. As a consequence, the bronchioles become irritableand hyperresponsive, rendering the individual more sensitive to avariety of inhaled irritants other than the allergen.

[0006] Rhinitis, allergic conjunctivitis and atopic dermatitis areinflammations of the nasal mucosa, eyes and skin, respectively, oftendue to allergens such as pollen, dust or other airborne substances.

[0007] Anaphylactic shock, the most severe form of allergy, is a medicalemergency. It is often severe and can sometimes provoke a fatal systemicreaction in a previously sensitized human or animal upon exposure to aspecific antigen, such as nuts, wasp or bee venom or penicillin.Anaphylactic shock is characterized by respiratory symptoms, fainting,itching, urticaria, swelling of the throat or other mucous membranesand/or a sudden decline in blood pressure. Symptoms of anaphylacticshock include dizziness, loss of consciousness, laboured breathing,swelling of the tongue, blueness of the skin, bronchospasm, low bloodpressure, and death. Anaphylactic reactions can also cause modificationof heart function and are associated with an increase in neutrophilinflux into the heart tissue (Turesin et al, 2000). This inflammation ofthe heart is effected by the leukocyte adhesion cascade.

[0008] For the most part, the available drugs for treating asthma blockor neutralize the effects of the release of inflammatory mediators suchas histamine, prostaglandins, leukotrienes, platelet activating factor(PAF) and chemotactic factors for monocytes, neutrophils andeosinophils. These mediators activate smooth muscle, causing bronchialconstriction, and act on adjacent immune, epithelial and endothelialcells to facilitate and amplify the inflammatory response. The drugscurrently available for treating asthma include the beta-adrenergics,corticosteroids, anti-leukotrienes, anti-cholinergics, xanthines and,less frequently non-steroidal anti-inflammatory drugs, (NSAIDS).

[0009] These drugs, when used properly, provide good management ofasthma. A significant number of asthma sufferers, however are poorlycontrolled and a need for improved anti-asthmatic drug therapy remains.

[0010] Anti-inflammatory peptides derived from the salivary gland havebeen described previously. These include submandibular gland peptide-T(SGP-T), which inhibits lipopolysaccharide-induced hypotension at dosesas low as 1 μg/kg (Mathison et al., (1997) Amer. J. Physiol., v. 273, p.R1017) and has also been shown to inhibit systemic and intestinal Type 1hypersensitivity reactions (Befus et al., (1997) Int. Arch. AllergyImmunol., v. 113, p. 337; Mathison et al, (1997) supra and Proc. WestPharmacol. Soc., v. 40, p. 73). Further studies demonstrated thattripeptide or larger fragments of SGP-T also have significantanti-inflammatory activity (International Patent Application WO98/06742).

SUMMARY OF THE INVENTION

[0011] The present invention provides novel peptides which are potentinhibitors of inflammatory reactions in mammals.

[0012] The di- and tripeptides of the invention have been shown toinhibit inflammatory reactions due to a wide variety of causes and canbe used to treat inflammatory reactions and disorders involving aninflammatory reaction in general, in human and non-human mammals. Thepeptides have human and veterinary pharmaceutical uses. They may beformulated as pharmaceutical compositions or used as food additives.

[0013] In accordance with one embodiment, the invention provides amethod of inhibiting an inflammatory reaction in a mammal comprisingadministering to the mammal an effective amount of a peptide of theformula:

[0014] X¹-X²-X³ wherein X¹ is selected from the group consisting of2-amino-hexanoic acid; 2-amino-heptanoic acid; 2-amino-octanoic acid;cyclohexyl-substituted 2-amino-ethanoic acid, 2-amino-propanoic acid or2-amino-butanoic acid and methionine;

[0015] X² is an acidic amino acid; and

[0016] X³ is an aliphatic amino acid; or of a peptide of the formula:

[0017] X⁴—X⁵ wherein X⁴ is an aromatic or aliphatic amino acid; and

[0018] X⁵ is an acidic amino acid.

[0019] In accordance with a further embodiment, the invention provides amethod of treating asthma in a mammal comprising administering to themammal an effective amount of a peptide of the formula:

[0020] X¹-X²-X³ wherein X¹ is selected from the group consisting of2-amino-hexanoic acid; 2-amino-heptanoic acid; 2-amino-octanoic acid;cyclohexyl-substituted 2-amino-ethanoic acid, 2-amino-propanoic acid or2-amino-butanoic acid and methionine;

[0021] X² is an acidic amino acid; and

[0022] X³ is an aliphatic amino acid; or of a peptide of the formula:

[0023] X⁴—X⁵ wherein X⁴ is an aromatic or aliphatic amino acid; and

[0024] X⁵ is an acidic amino acid.

[0025] In accordance with a further embodiment, the invention provides apeptide of the formula: X¹—X²—X³ wherein

[0026] X¹-X²-X³ wherein X¹ is selected from the group consisting of2-amino-hexanoic acid; 2-amino-heptanoic acid; 2-amino-octanoic acid;cyclohexyl-substituted 2-amino-ethanoic acid, 2-amino-propanoic acid or2-amino-butanoic acid and methionine;

[0027] X² is an acidic amino acid; and

[0028] X³ is an aliphatic amino acid.

[0029] In accordance with a further embodiment, the invention provides apeptide of the formula: X⁴—X⁵ wherein X⁴ is an aromatic or aliphaticamino acid; and

[0030] X⁵ is an acidic amino acid.

[0031] In accordance with a further embodiment, the invention provides apharmaceutical composition comprising a peptide as described above and apharmaceutically acceptable carrier.

[0032] In accordance with a further embodiment, the invention providesuse of a peptide as described above for the preparation of a medicamentfor inhibiting an inflammatory reaction or for treating asthma.

SUMMARY OF THE DRAWINGS

[0033] Certain embodiments of the invention are described, referencebeing made to the accompanying drawings, wherein:

[0034]FIG. 1A shows the effect of aerosol feG treatment onantigen-induced airways bronchoconstriction in sheep, expressed as %change in SR_(L), over time (hours) after antigen challenge. Values areexpressed as mean±SEM for two animals.

[0035]FIG. 1B shows the effect of aerosol feG on antigen-induced airwayshyperreponsiveness in sheep, expressed as the number of breaths (BreathUnits) required to increase airways resistance by 400% (PC₄₀₀), before(Baseline) and at 24 h Post-antigen challenge (24 h P-Ag). Values areexpressed as mean±SEM for two animals.

[0036]FIGS. 2A and 2B show the effect of intravenous treatment with feGon antigen-induced airway responses in sheep expressed as in FIGS. 1Aand 1B.

[0037]FIGS. 3A and 3B show the effect of two oral pre-challengetreatments with feG on antigen-induced airway responses in sheepexpressed as in FIGS. 1A and 1B.

[0038]FIGS. 4A and 4B show the effect of aerosol treatment with (cha)eGon antigen-induced airway responses in sheep expressed as in FIGS. 1Aand 1B.

[0039]FIGS. 5A and 5B show the effect of intravenous treatment with(cha)eG on antigen-induced airway responses in sheep expressed as inFIGS. 1A and 1B.

[0040]FIGS. 6A and 6B show the effect of two oral pre-challengetreatments with (cha)eG on antigen-induced airway responses in sheepexpressed as in FIGS. 1A and 1B.

[0041]FIGS. 7A and 7B show the effect of aerosol treatment with (Cha)EGon antigen-induced airway responses in sheep expressed as in FIGS. 1Aand 1B.

[0042]FIGS. 8A and 8B show the effect of intravenous treatment with(Cha)EG on antigen-induced airway responses in sheep expressed as inFIGS. 1A and 1B.

[0043]FIGS. 9A, 9B and 9C show the inhibitory effect of peptides of theinvention on immunoglobulin-mediated vascular permeability in rats,expressed as % decrease in leak.

[0044]FIG. 10 shows the inhibitory effect of peptides of the inventionon active cutaneous anaphylaxis in rats.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The amino acids which are components of normal mammalian proteinsare referred to herein by the conventional one letter or three lettercodes. When the one letter code is used, L-isomeric forms of amino acidsare identified by capital letters and D-forms by lower case letters,e.g. L-phenylalanine: F; D-phenylalanine:f. Reference to an amino acidwithout explicit indication of isomeric-form refers to the L form.

[0046] Other amino acids referred to herein are identified as follows:Amino Acid Abbreviation Formula β alanine β Al (NH₂)—CH₂—CH₂—COOH γamino butyric acid γ Abu (NH₂)—(CH₂)—₃—COOH L-cyclohexylalanine ChaC6H11—CH₂—CH(NH₂)—COOH D-cyclohexylalanine cha homoserine HseHO—(CH₂)₂—CH(NH₂)—COOH norleucine Nle CH_(3—)(CH₂)₃—CH(NH₂)—COOHnorvaline Nval CH₃—(CH₂)₂—CH(NH₂)—COOH phenyl glycine PhgC6H5—CH(NH₂)—COOH sarcosine Sar NH(CH₃)—CH₂—COOH

[0047] An “acidic amino acid” is an amino acid which has a side chaincontaining one or more carboxyl groups.

[0048] Beginning with the peptide FEG, which has been shown to haveanti-inflammatory activity, a number of analogues of the peptide havebeen synthesised, substituting one or more amino acids with a differentamino acid, in either D or L form, and the anti-inflammatory activity ofthese analogues has been examined. Based on these studies, and theunexpected results of various substitutions, the invention provides newgroups of peptides which are potent modulators of inflammatory reactionsin mammals.

[0049] Although earlier studies suggested that the N-terminal amino acidof FEG analogues had to be aromatic for activity, it has been foundunexpectedly that the N-terminal amino acid may be aliphatic. It hasfurther been found unexpectedly that a dipeptide may be sufficient foranti-inflammatory activity.

[0050] The invention provides anti-inflammatory peptides of the formula:X¹—X²—X³ wherein X¹ is selected from the group consisting of2-amino-hexanoic acid; 2-amino-heptanoic acid; 2-amino-octanoic acid;cyclohexyl-substituted 2-amino-ethanoic acid, 2-amino-propanoic acid or2-amino-butanoic acid, and methionine; X² is an acidic amino acid; andX³ is an aliphatic amino acid.

[0051] Of the cyclohexyl-substituted amino acids,2-amino-2-cyclohexyl-ethanoic acid, 2-amino-3-cyclohexyl-propanoic acid,2-amino-3-cyclohexyl-butanoic acid and 2-amino-4-cyclohexyl-butanoicacid are preferred.

[0052] Especially preferred amino acids for position X¹ are2-amino-3-cyclohexyl-propanoic acid (cyclohexylalanine),2-amino-hexanoic acid (norleucine) and methionine.

[0053] X² is preferably glutamic acid and X³ is preferably glycine,methionine, isoleucine, alanine, β-alanine, sarcosine or γ amino butyricacid.

[0054] The invention further provides anti-inflammatory peptides of theformula: X⁴—X⁵ wherein X⁴ is an aromatic or aliphatic amino acid; and X⁵is an acidic amino acid.

[0055] X⁴ is preferably phenylalanine, norleucine, cyclohexylalanine,methionine or tyrosine and X⁵ is preferably glutamic acid.

[0056] One or more of the amino acids of the peptides of the inventionmay be D-amino acids.

[0057] In a further embodiment, and by analogy with previous findings onamidated FEG and feG, the tripeptides and dipeptides of the inventionmay be used in amidated form, i.e. the C-terminal amino acid of thepeptide is amidated. Such amidated peptides may be more stable in themammalian body than the carboxy terminal peptides.

[0058] In a further embodiment, in the peptides of the invention, theamide of the N-terminal amino acid may be substituted with a straightchain lower alkyl group of 1 to 3 carbons. Where methionine is theN-terminal amino acid, it may be thio-substituted. Cyclohexyl-containingpeptides may be lower alkyl substituted on the cyclohexyl ring. Loweralkyl substitution is preferably methyl substitution.

[0059] An inflammatory reaction in a mammal is a protective responseagainst a variety of damaging or potentially damaging agents. Suchagents include allergens, toxins, bacteria, viruses, parasites andfungi.

[0060] Inflammatory reactions are associated with increased vascularpermeability and blood flow, migration of leukocytes to the site of theinflammatory reaction, and release of vasodilatory, cytotoxic and tissuerepair molecules.

[0061] Depending on the tissue or organ involved, an inflammatoryreaction will lead to different symptoms. For example, an inflammatoryreaction in the lungs may lead to pulmonary inflammation,bronchoconstriction and airway obstruction.

[0062] Inflammatory reactions are involved in the symptomatology ofasthma, whether triggered by external agents such as allergens, as inextrinsic asthma, or by non-allergen factors, as in intrinsic asthma.

[0063] Inflammatory response syndrome (IRS) involves a severe andwidespread inflammatory reaction affecting multiple organs and tissue.IRS occurs as a result of extensive tissue damage and necrosis or theinvasion of microorganisms, with the release of chemical mediators orcellular by-products such as the cytokines, lipid metabolites andautocoids. These mediators can be released as a result of tissues andcells affected by infections, shock (endotoxemia, blood loss, blunttrauma), hypoxemia, radiation, burns, organ transplants, graftrejections. The inflammatory IRS is primarily responsible for thedevelopment of organ dysfunctions, such as acute lung injury, acuterespiratory distress syndrome (ARDS), damage to gastrointestinaldysfunction (ileus, changes in permeability, pancreatitis likeproblems), and dysfunctions of the kidney, heart, liver and brain.

[0064] An acute and overwhelming reaction to an allergen may lead toanaphylactic shock, where many organs and systems of the body areaffected by the inflammatory reaction.

[0065] Exposure to an allergen can cause an inflammatory reaction in theintestines which, in severe cases, can cause intestinal anaphylaxis oreven systemic anaphylaxis. Cardiac inflammation may result from ischemiaor as a component of reperfusion injury.

[0066] Inflammatory reactions also include endotoxic reactions,immunological reactions and hypersensitivity reactions, including TypeI, Type II and Type III hypersensitivity reactions.

[0067] Inflammatory reactions also include endotoxic reactions tolipopolysaccharide components of gram negative bacteria.

[0068] Type I hypersensitivity reactions or allergic reactions aretypically immunoglobulin E-mediated and occur in many allergicdisorders, including allergic rhinitis, allergic conjunctivitis, atopicdermatitis, extrinsic asthma, and some cases of urticaria,gastrointestinal reactions to food and systemic anaphylaxis.

[0069] Type II and Type III hypersensitivity reactions are generallymediated by antibodies other than IgE, and can involve immunoglobulinG-mediated reactions, as well as those using other immunoglobulins. TypeII reactions are cytotoxic reactions resulting when antibody reacts withantigenic components of a cell or tissue elements or with antigen orhapten that is coupled to a cell or tissue. The antigen-antibodyreaction may activate certain cytotoxic cells (e.g. killer T cells ormacrophages) to produce antibody-dependent cell-mediated cytotoxicity.Type II reactions are involved in disorders including hemolyticanaemias, antibody-induced thrombocytopenic purpura, leukopenia,pemphigus, pemphigoid, Goodpasture's syndrome, pernicious anaemia,incompatible blood transfusions, neonatal thrombocytopenia andmultisystem hypersensitivity diseases such as scleroderma. Type IIIreactions are immune complex (IC) reactions resulting from deposition ofsoluble circulating antigen-antibody ICs in vessels or tissue, and areinvolved in disorders including serum sickness due to serum, drugs orviral hepatitis antigen, scleroderma rheumatoid arthritis,polyarteritis, cryoglobulinemia, hypersensitivity pneumonitis,bronchopulmonary aspergillosis, acute glomerulonephritis, chronicmembrane proliferative glomerulonephritis and related renal diseases.

[0070] The results described herein indicate that the compounds of theinvention are effective in inhibiting both inflammatory reactionsinvolving Type I hypersensitivity reactions (e.g. allergic asthma,intestinal response to antigen) and inflammatory reactions involvingType III hypersensitivity reactions (e.g. vascular permeability andcutaneous anaphylaxis).

[0071] In any disorder involving an inflammatory reaction, the subject'ssymptoms can be ameliorated and the disorder treated by administering ananti-inflammatory agent to the subject to inhibit the reaction. Althoughit is not yet known by what mechanism the peptides of the inventioninterfere with inflammatory reactions, the peptides of the invention maybe used to inhibit inflammatory reactions generally. It is clear fromthe studies described herein that they are effective to inhibitinflammatory reactions involving a wide variety of causes, includingallergen-induced and non-allergen-induced asthma, allergen-induced andfood intolerance-related intestinal disorders, anaphylactic shock,endotoxic reactions and inflammatory response syndrome.

[0072] The compounds of the invention may also be used as veterinarypharmaceuticals, for the treatment of a variety of diseases andconditions common to mammals other than humans. All animals canexperience Types I, II and III hypersensitivity reactions, immunologicalreactions, and the other types of inflammatory reactions describedherein.

[0073] One of the most common conditions affecting household pets,including cats and dogs, is allergy, which can affect all major organsystems. These allergies are characterized by itching of the skin,either localized or generalized, hyper-responsive (over-reactive)airways, coughing, sneezing and/or wheezing and discharge from noseand/or ears or vomiting or diarrhea. Non-human mammals which may betreated with the methods and compositions of the invention include dogs,cats, mice, rats, rabbits, horses, cows, sheep and other domestic petsand farm animals.

[0074] The invention therefore provides new pharmaceutical compositionsand methods for inhibiting an inflammatory reaction in human andnon-human mammals, comprising administering to the mammal an effectiveamount of a peptide as described herein. These compositions and methodscan be employed to treat a wide variety of disorders involving aninflammatory reaction, including asthma, rhinitis, conjunctivitis,atopic dermatitis, intestinal anaphylaxis, allergen-induced and foodintolerance related intestinal disorders and systemic allergicreactions.

[0075] The peptides of the invention can also be employed to treatpulmonary inflammation and bronchoconstriction and immunologicalreactions and to inhibit chemotaxis of inflammatory cells, includingneutrophils, into an inflammatory site.

[0076] An “effective amount” means an amount sufficient to produce asignificant inhibition of an inflammatory reaction, as evidenced, forexample, by an observable amelioration of one or more symptoms producedby the inflammatory reaction or by a reduction in one or more of theabove-described indicia of inflammatory reactions, such as increasedvascular permeability, leukocyte migration, etc.

[0077] Peptides in accordance with the invention may be prepared by anysuitable peptide synthetic method, as are known to those of skill in theart.

[0078] Chemical synthesis may be employed; for example standard solidphase peptide synthetic techniques may be used. In standard solid phasepeptide synthesis, peptides of varying length can be prepared usingcommercially available equipment. This equipment can be obtained, forexample, from Applied Biosystems (Foster City, Calif.). The reactionconditions in peptide synthesis are optimized to prevent isomerizationof stereochemical centres, to prevent side reactions and to obtain highyields. The peptides are synthesized using standard automated protocols,using t-butoxycarbonyl-alpha-amino acids, and following themanufacture's instructions for blocking interfering groups, protectingthe amino acid to be reacted, coupling, deprotecting and capping ofunreacted residues. The solid support is generally based on apolystyrene resin, the resin acting both as a support for the growingpeptide chain, and as a protective group for the carboxy terminus.Cleavage from the resin yields the free carboxylic acid. Peptides arepurified by HPLC techniques, for example on a preparative C18 reversephase column, using acetonitrile gradients in 0.1% trifluoroacetic acid,followed by vacuum drying. The peptides of the invention can also beproduced by liquid phase peptide chemistry.

[0079] Peptides may also be produced by recombinant synthesis. A DNAsequence encoding the desired peptide is prepared and subcloned into anexpression plasmid DNA. Suitable mammalian expression plasmids includepRC/CMV from Invitrogen Inc. The gene construct is expressed in asuitable cell line, such as a Cos or CHO cell line and the expressedpeptide is extracted and purified by conventional methods. Suitablemethods for recombinant synthesis of peptides are described in Sambrooket al., (1989), “Molecular Cloning” Cold Spring Harbor, Lab. Press, ColdSpring Harbor, N.Y. Derivatives of a peptide may be prepared by similarsynthetic methods. Examples of side chain modifications contemplated bythe present invention include modification of amino groups such as byreductive alkylation by reaction with an aldehyde followed by reductionwith NaBH₄; amidation with methylacetimidate; acetylation with aceticanhydride; carbamylation of amino groups with 2, 4, 6, trinitrobenzenesulfonic acid (TNBS); alkylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5′-phosphate followed by reduction with NaBH₄.

[0080] The guanidino group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

[0081] The carboxyl group may be modified by carbodiimide activation viaacylisourea formation followed by subsequent derivatisation, forexample, to a corresponding amide.

[0082] Examples of incorporating unnatural amino acids and derivativesduring peptide synthesis include, but are not limited to, use ofnorleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoicacid, 6-aminohexanoic acid-, 5-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers or amino acids.

[0083] Synthesized peptides may be screened for their anti-inflammatoryactivity by one of the assay systems described herein. The assayselected will depend on the biological activity of interest in eachcase. For example, peptides may be screened for their effectiveness ininhibiting vascular permeability changes by the method described inExample 3 or for their effectiveness in inhibiting antigen-inducedairway bronchoconstriction, as described in Example 2.

[0084] The peptides of the invention may be administered therapeuticallyby injection or by oral, nasal, buccal, sub-lingual, rectal, vaginal,transdermal or ocular routes in a variety of formations, as in known tothose in the art.

[0085] For oral administration, various techniques can be used toimprove stability, based for example on chemical modification,formulation and use of protease inhibitors. Stability can be improved ifsynthetic amino acids are used, such as peptides or betidamino acids, orif metabolically stable analogues are prepared.

[0086] Formulation may be, for example, in water/oil emulsion or inliposomes for improved stability. Oral administration of peptides may beaccompanied by protease inhibitors such as aprotinin, soybean trypsininhibitor or FK-448, to provide protection for the peptide. Suitablemethods for preparation of oral formulations of peptide drugs have beendescribed, for example by Saffran et al., (1979), Can. J. Biochem., v.57, pp. 548-553; Lundin et al., (1986), Life Sci., v. 38, pp. 703-709,and Vilhardt et al., (1986), Gen. Pharmacol., v. 17, pp. 481-483.

[0087] Due to the high surface area and extensive vascular network, thenasal cavity provides a good site for absorption of both lipophilic andhydrophilic drugs, especially when coadministered with absorptionenhancers. The nasal absorption of peptide-based drugs can be improvedby using aminoboronic acid derivatives, amastatin, and other enzymeinhibitors as absorption enhancers and by using surfactants such assodium glycolate, as described in Amidon et al., (1994), Ann. Rev.Pharmacol. Toxicol., v. 34, pp. 321-341. The transdermal route providesgood control of delivery and maintenance of the therapeutic level ofdrug over a prolonged period of time, Amidon et al., (1994), Ann. Rev.Pharmacol. Toxicol., v. 34, pp. 321-341 and Choi et al., (1990), Pharm.Res., v. 7, pp. 1099-1106. A means of increasing skin permeability isdesirable, to provide for systemic access of peptides. For example,iontophoresis can be used as an active driving force for chargedpeptides or chemical enhancers such as the nonionic surfactantn-decylmethyl sulfoxide (NDMS) can be used.

[0088] Peptides may also be conjugated with water soluble polymers suchas polyethlene glycol, dextran or albumin or incorporated into drugdelivery systems such as polymeric matrices to increase plasmahalf-life.

[0089] More generally, formulations suitable for particular modes ofadministration of peptides are described, for example, in Remington'sPharmaceutical Sciences (Ed. Gennaro, A. R., (2000), 20^(th) Edition,Williams & Wilkins Pa., U.S.A.).

[0090] The particular dosage required in a given subject can bedetermined by the attending physician. A starting dosage in the range of1 μg-1000 μg peptide/kg body weight can be employed, with adjustment ofthe dosage based on the response of a particular subject, as understoodby those of ordinary skill in the art.

[0091] The peptides of the invention may also be formulated as foodsupplements by their addition to food products or beverage products. Theuse of peptides as food additives and their incorporation into food orbeverage products is well known to those of skill in the food processingart. Where the peptides contain only natural amino acids, these productsare attractive to those who favour natural medicines and natural healthproducts.

EXAMPLES

[0092] The examples are described for the purposes of illustration andare not intended to limit the scope of the invention.

[0093] Methods of chemistry, protein and peptide biochemistry andimmunology referred to but not explicitly described in this disclosureand examples are reported in the scientific literature and are wellknown to those skilled in the art.

Example 1

[0094] Effects of Peptides on Antigen-induced Contraction of SensitizedRat Ileum.

[0095] For this in vitro motility study, peptides were prepared byconventional methods at the Protein Synthesis Facilities, University ofCalgary, Calgary, Alberta, or by Core Laboratories, Queen's University,Kingston, Ontario, and examined for their inhibitory effects on ananaphylactic reaction provoked by antigen on intestinal segmentsisolated from ovalbumin sensitized rats. The procedures described byMathison et al. (1997), (supra) were followed with slight modifications.Sprague-Dawley rats were sensitized to 1 mg ovalbumin (OA) and 50 ngpertussis toxin (Sigma Chemical, St. Louis, Mo.) (Kosckea et al, 1994,Am. J. Physiol., v. 267, pG745). Four to six weeks followingsensitization, the terminal ileum was excised and 2 cm sections weremounted in 20 ml organ baths under 0.75 g of tension and the isometricforce generated by OA and urecholine (Frosst, Kirkland, QC) was measuredusing a Grass Force Displacement Transducer FT03 (Quincy, Mass.). Thetissues were washed several times in Krebs and allowed to equilibratefor 15 minutes. Anti-anaphylactic properties of the peptides weredetermined by adding 10 μg of peptide to a bath and incubating for 10min. Tissue segments were washed, the baseline reestablished, and thenchallenged with 1 mg of the OA antigen. OA contractile response wasmeasured at peak contraction. Tissues were washed and peak contractileresponse obtained by adding urecholine to a concentration of 10⁻⁵M. Themucosa was then scraped from the tissue, the mass of the remainingmuscle determined, and the tension calculated in gram force per gram wettissue. Results were expressed as the ratio of OA induced contractileresponse to urecholine induced contractile response. To obtain therelative activity the OA/URE ratio for each peptide was expressed as apercent of control.

[0096] The results are shown in Table 1. The addition of antigen (OA)induced a slow tonic contraction of the intestinal (ileal) segmentswhich peaked within 2 to 3 minutes before slowly receding to thebaseline tension over 4 to 5 minutes. Urecholine (URE) produced a rapidtonic contraction that reached its maximum in 10 to 15 seconds. Thetripeptide analogues did not alter the contractile response to URE (notshown), which allowed the use of the OA/URE ratio as a measure of theanti-anaphylactic responses of the peptide. The OA/URE ratio inuntreated tissues was 0.26±0.02, which indicates that the sensitizingantigen caused a contractile response that was 26% of that ofurecholine.

Example 2

[0097] Treatment of Asthmatic Reactions in Sheep

[0098] Animal Preparation: Experiments were performed as described byAbraham et al. (2000), Am. J. Respir. Crit. Care Med., v. 162, p. 603.Allergic sheep, weighing 27 to 50 kg, had previously been shown to havea natural allergic cutaneous reaction to 1:1000 or 1:10,000 dilutions ofAscaris suum extract (Greer Diagnostics, Lenoir, N.C.) and to developboth early and late bronchial responses (“dual responders”) to inhaledAscaris suum antigen (Abraham et al, 1983, Am. Rev. Respir. Dis., v.128, p. 839). The sheep were conscious and restrained in a modifiedshopping cart in the prone position with their heads immobilized.Topical anesthesia (2% lidocaine) was applied to the nasal passages, anda balloon catheter was advanced through one nostril into the loweresophagus. The animals were then intubated with a cuffed endotrachealtube through the other nostril with a flexible fiberoptic bronchoscopeas a guide. All protocols were approved by the Mount Sinai MedicalCenter Animal Research Committee, which is responsible for assuring thehumane care and use of experimental animals.

[0099] Airway Mechanics: Breath-by-breath determination of meanpulmonary flow resistance (RL) was measured with the esophageal balloontechnique previously described (Abraham et al, (1994), J. Clin. Invest.,v. 93, p. 776 and (1997), Am. J. Respir. Crit. Care Med., v. 156, p.696). The mean of at least 5 breaths, free of swallowing artifact, wereused to obtain RL in cm H2O/L/s. Immediately after the measurement ofRL, thoracic gas volume (Vtg) was measured in a constant-volume bodyplethysmograph to obtain specific lung resistance (SRL=RL×Vtg) in cmH2O.s1.

[0100] Aerosol Delivery Systems: All aerosols were generated using adisposable medical nebulizer (Raindrop; Puritan Bennett, Lenexa, Kans.)that provided an aerosol with a mass median aerodynamic diameter of 3.2μm as determined by an Andersen cascade impactor. The nebulizer wasconnected to a dosimeter system, consisting of a solenoid valve and asource of compressed air (20 psi). The output of the nebulizer wasdirected into a plastic T-piece, one end of which was connected to theinspiratory port of a piston respirator (Harvard Apparatus, Mills,Mass.). The solenoid valve was activated for 1 s at the beginning of theinspiratory cycle of the respirator. Aerosols were delivered at a tidalvolume of 500 ml and a rate of 20 breaths/min as previously described(Abraham et al, 1994; 1997 supra).

[0101] Concentration Response Curves to Carbachol Aerosol: Airwayresponsiveness was determined from cumulative concentration responsecurves to carbachol inhaled as previously described. SR_(L) was measuredimmediately after inhalation of phosphate-buffered saline (PBS.) andafter each consecutive administration of 10 breaths of increasingconcentrations of carbachol (0.25, 0.5, 1.0, 2.0, and 4.0% wt/vol PBS).The provocation test was discontinued when SR_(L) increased over 400%from the post-PBS value or after the highest carbachol concentration hadbeen administered. The cumulative carbachol concentration (in breathunits [BU]) that increased SR_(L) by 400% over the post-PBS value(PC400) was calculated by interpolation from the dose-response curve.One BU was defined as one breath of a 1% wt/vol carbachol aerosolsolution (Abraham et al, 1994; 1997 supra).

[0102] Antigen Challenge: A. suum extract (Greer Diagnostics, Lenoir,N.C.) was diluted with PBS to a concentration of 82,000 protein nitrogenunits/ml and delivered as an aerosol (20 breaths/min×20 min).

[0103] Carbamylcholine (Carbachol; Sigma Chemical Co., St. Louis, Mo.)was dissolved in PBS at concentrations of 0.25, 0.50, 1.0, 2.0, and 4.0%wt/vol and delivered as an aerosol. Peptides were dissolved in 0.9%saline prepared in 3 to 5 ml total volume depending on the route ofadministration (aerosol, oral or intravenously) for the study.

[0104] Experimental Trials: Each sheep was subjected to a control trialin which a placebo (PBS without additive) was used as the testpretreatment before allergen challenge with Ascaris suum antigen.Subsequently, the sheep were pretreated, prior to allergen challenge,with a test peptide (prepared by Core Laboratories) in sterile,endotoxin-free PBS, as follows:

[0105] (i) feG (30 mg/sheep) was given by aerosol 20 min. beforechallenge with antigen. FIG. 1A shows bronchoconstriction and FIG. 1Bshows airway hyperresponsiveness, after antigen challenge.

[0106] (ii) feG (1 mg/kg) was given intravenously 20 min. before antigenchallenge. FIG. 2A shows bronchoconstriction and FIG. 2B shows airwayhyperresponsiveness, after antigen challenge.

[0107] (iii) feG was administered orally, at a dose of 2 mg/kg, at 24 h.and 30 min. before antigen challenge. FIG. 3A shows bronchoconstrictionand FIG. 3B shows airway hyperresponsiveness, after antigen challenge.

[0108] (iv) (cha)eG (30 mg/sheep) was given by aerosol 20 min. beforeantigen challenge. FIG. 4A shows bronchoconstriction and FIG. 4B showsairway hyperresponsiveness, after antigen challenge.

[0109] (v) (cha)eG (1 mg/kg) was given intravenously 20 min beforeantigen challenge. FIG. 5A shows bronchoconstriction and FIG. 5B showsairway hyperresponsiveness, after antigen challenge.

[0110] (vi) (cha)eG was administered orally, at a dose of 2 mg/kg, at 24h and 30 min before antigen challenge. FIG. 6A shows bronchoconstrictionand FIG. 6B shows airway hyperresponsiveness, after antigen challenge.

[0111] (vii) (Cha)EG (30 mg/sheep) was given by aerosol 20 min beforechallenge with antigen FIG. 7A shows bronchoconstriction and FIG. 7Bshows airway hyperresponsiveness, after antigen challenge.

[0112] (viii) (Cha)EG (1 mg/kg) was given intravenously 20 min beforeantigen challenge. FIG. 8A shows bronchoconstriction and FIG. 8B showsairway hyperresponsiveness, after antigen challenge.

[0113] Activity of analogue peptides was compared with activity of feG.

[0114] Sheep that had received a total dose of 30 mg of feG by aerosolinhalation, 20 min before antigen challenge, exhibited attenuation of78% and 89%, respectively, of the early (0-3 h) and the late (6-8 h)bronchoconstriction provoked by aerosol administration of Ascaris suumantigen. This peptide also reduced the increase in airwayhyperresponsiveness to methacholine with a 1.9-fold lower dose producinga PC₄₀₀ response.

[0115] Sheep that had received a dose of 1 mg/kg of feG givenintravenously, 20 min before antigen challenge, exhibited an attenuationof 72% of the late (6-8 h) bronchoconstriction provoked by aerosoladministration of Ascaris suum antigen. The early bronchoconstrictorphase was not affected. Intravenous treatment with feG also reduced theincrease in airways hyperresponsiveness to methacholine with a 2.2-foldlower dose producing a PC₄₀₀ response.

[0116] Sheep that had received feG orally, in two doses of 2 mg/kg, 24 hand 30 min before intravenous antigen challenge, exhibited anattenuation of 69% of the late (6-8 h) bronchoconstriction provoked byaerosol administration of Ascaris summ antigen. The earlybronchoconstrictor phase was not affected. Intravenous treatment withfeG also reduced the increase in airways hyperresponsiveness tomethacholine with a 2.1-fold lower dose producing a PC₄₀₀ response.

[0117] Sheep that had received a total dose of 30 mg of (cha)eG byaerosol, 20 min before antigen challenge, exhibited attenuation of 64%and 88%, respectively, of the early (0-3 h) and the late (6-8 h)bronchoconstriction provoked by aerosol administration of Ascaris summantigen. Aerosol treatment with (cha)eG also reduced the increase inairways hyperresponsiveness to methacholine with a 2.8-fold lower doseproducing a PC₄₀₀ response.

[0118] Sheep that had received a dose of 1 mg/kg of (cha)eG givenintravenously, 20 min before antigen challenge, exhibited attenuation of58% and 43%, respectively, of the early (0-3 h) and the late (6-8 h)bronchoconstriction provoked by aerosol administration of Ascaris summantigen. This peptide also reduced the increase in airwayshyperresponsiveness to methacholine with a 1.7-fold lower dose producinga PC₄₀₀ response.

[0119] Sheep that had received (cha)eG orally in two doses of 2 mg/kg,24 h and 30 min before antigen challenge intravenously, exhibited anattenuation of 58% and 43%, respectively, of the early (0-3 h) and thelate (6-8 h) bronchoconstriction provoked by aerosol administration ofAscaris summ antigen. When administered orally (cha)eG reduced theincrease in airway hyperresponsiveness to methacholine with a 1.7-foldlower dose producing a PC₄₀₀ response.

[0120] Sheep that received a total dose of 30 mg of (Cha)EG by aerosolinhalation, 20 min before antigen challenge, exhibited attenuation of52% and 84%, respectively, of the early (0-3 h) and the late (6-8 h)bronchoconstriction provoked by aerosol administration of Ascaris summantigen. This peptide, (Cha)EG, reduced the increase in airwayhyperresponsiveness to methacholine with a 1.9-fold lower dose producinga PC₄₀₀ response.

[0121] Sheep that received a dose of 1 mg/kg of (Cha)EG intravenously,20 min before antigen challenge, exhibited attenuation of 62% and 67%,respectively, of the early (0-3 h) and the late (6-8 h)bronchoconstriction provoked by aerosol administration of Ascaris summantigen. This peptide, (Cha)EG, reduced the increase in airwayhyperresponsiveness to methacholine with a 3.2-fold lower dose producinga PC₄₀₀ response.

Example 3

[0122] Inhibition of Antibody-Mediated Vascular Permeability and ActiveCutaneous Anaphylaxis in Rats

[0123] For this in vivo study, peptides were prepared by conventional byCore Laboratories, Queen's University, Kingston, Ontario, and examinedfor their inhibitory effects on an vascular permeability elicited byIgG- or IgE-antibodies and on active cutaneous anaphylactic (ACA)reactions. The IgG- or IgE-antibody reactions were studies in normal,unsensitized Sprague-Dawley rats anaethetized with sodium pentobarbital(65 mg/kg). The backs of the animals were shaved, and a 2×4 grid wasdrawn on the back in black ink and in the middle of each square 50 μl ofdifferent concentrations of peptide (10-12 to 10-7 moles/l) wereinjected intradermally. Saline was used as a control. For IgG-mediatedreactions rats were injected intravenously, via the penile vein, with 10mg/kg of ovalbumin and 20 mg/kg of Evans blue, ten min later the IgGantibody (mouse anti-ovalbumin) and after 3 h the area of the vascularleak, visualized by the blue spots, was calculated with the formula forthe surface area of an ellipse. Similar procedures were applied forstudying IgE-mediated vascular leak in that rats were injectedintravenously with 20 mg/kg of Evans blue, and 10 min later an amonoclonal antibody against the heavy chain of the rat IgE receptor wasinjected intradermally.

[0124] ACA reactions were elicited by antigen injections into the dorsalskin of ovalbumin sensitized rats. Sprague-Dawley rats were sensitizedto 1 mg ovalbumin (OA) and 50 ng pertussis toxin (Sigma Chemical, St.Louis, Mo.) (Kosckea et al, 1994). Four to six weeks followingsensitisation, anti-OA IgE antibody titer was determined via passivecutaneous anaphylaxis and rats with an antibody titre of 1:32 were used.To perform ACA rats were anaethetized with sodium pentobarbital (65mg/kg) and their backs shaved. A 2×4 grid was drawn on the back in blackink and in the middle of each square different concentrations of peptide(10-12 to 10-6 moles) in 50 μl were injected intradermally. Saline wasused as a control. The animals were then injected, via the penile vein,with Evans blue (20 mg/kg), a dye that binds to albumin. Ten minutesafter the first intradermal injection 50 μl of ovalbumin antigen (100μg/ml) was injected into the same site as the saline or peptide. Withina few minutes of the antigen injection into a control site, a blueing ofthe skin begins to develop as the Evans Blue-albumin complex moves outof the blood vessel consequent to antigen activation of mast cells.Thirty minutes after the injection of antigen the size of the histamineinduced wheal was measured using calipers.

[0125] The results are shown in FIGS. 9 and 10.

[0126] Both (cha)eG (FIG. 9A), (Cha)EG (FIG. 9B), and (Nle)EG (FIG. 9C)inhibited the increase in vascular permeability (% decrease in vascularleak) elicited by either IgG- or IgE-antibodies, or of both of theseantibodies. With all three peptides, the IgG-mediated vascular leak wasinhibited more than the IgE-mediated vascular leak. However, at lowconcentrations (10⁻¹¹M to 10⁻⁹M) (cha)eG provided a significantinhibition of the IgE-mediated vascular leak. The ACA reaction wasinhibited (reduced size of wheal) to a similar extent by both (Cha)EGand (cha)eG when injected at concentrations ranging from 10⁻¹¹M to 10⁻⁸M(FIG. 10).

Example 4

[0127] Inhibition by (Cha)EG, MEG, fe(Sar) and fe of Allergen InducedPulmonary Inflammation

[0128] The Brown Norway rat/ovalbumin sensitization model of allergicasthma was used. These rats are high IgE producers and are widely usedto study asthma because they develop an early and a late phasebronchoconstriction, as well as an increase in bronchialhyperresponsiveness.

[0129] Brown Norway rats (10-12 weeks old) were sensitized to ovalbumin(OA; Sigma Chemical Co. St. Louis, Mo.) with a 1 ml 0.9% saline ipinjection containing 10 μg OA, 150 mg A1 (OH)3 (ICN, Aurora, Ohio) and50 ng B. pertussis toxin (Sigma). Twenty-one days post sensitization,experiments were performed under light anaesthesia. Animals werechallenged with aerosolized OA (5%) and thirty minutes later they weregiven an oral treatment with either test peptide ((Cha)EG, MEG, fe(Sar)or fe: 250 μg/kg) or saline (controls).

[0130] Twenty-four hours after allergen challenge, the rats wereanaesthetised with pentobarbital (65 mg/kg) and cells were collectedfrom the lower respiratory tract by bronchoalveolar lavage (BAL). Theabdomen was opened, and the diaphragm was cut to relieve intrathoracicpressure. A tracheotomy was performed and a cannula inserted to thefirst bifurcation of the bronchioles. The bronchioles and alveoli werewashed 10 times with 5 ml of phosphate-buffered saline (PBS). The cellswere centrifuged at 200 g for 20 min and then resuspended in 1 ml ofPBS. The total number of cells were counted and differentials determinedwith May-Grunwald/Giesma stain.

[0131] The control rats responded to the aerosol challenge with 5%sensitizing antigen ovalbumin (OA) with a substantial pulmonaryinflammation at 24 h. The total number of cells recovered bybronchoalveolar lavage from the alveolar spaces increased from 3.5±1.8(×10⁶) cells in sensitized, unchallenged, rats to 25.0±3.2 (×10⁶) insensitized, antigen-challenged rats. (Cha)EG, MEG, fe(Sar) and fe (0.25mg/kg), when administered 30 min following allergen challenge ofsensitized rats, significantly reduced the total cell number recoveredby bronchoalveolar lavage by 50% to 80%, to 8.8±3.5 (×10⁶), 12.1±3.6(×10⁶), 5.1±2.6 (×10⁶) and 13.1±3.7 (×10⁶) respectively.

[0132] The present invention is not limited to the features of theembodiments described herein, but includes all variations andmodifications within the scope of the claims. TABLE 1 Structure ActivityRelationships for Tri- and Di-Amino Acid Peptides. Primary SubstitutionPeptide Avg SEM Significance Control 0.267 0.020 L-Phe FEG 0.125 0.040 *D-phe-D-glu feG 0.138 0.019 * D-phe-D-glu-D-ala fea 0.128 0.018 *D-tyr-D-glu yeG 0.157 0.010 * L-Phg (Phg)EG 0.136 0.023 * L-Trp WEG0.128 0.052 * L-Trp-L-Asp WDG 0.158 0.024 * NMePhe (NMeF)EG 0.1180.027 * L-Nle (Nle)EG 0.127 0.027 * L-Cha (Cha)EG 0.092 0.014 * L-MetMEG 0.121 0.027 * L-Met (position 3) FEM 0.081 0.016 * L-Ile (position3) FEI 0.161 0.018 * β-alanine (position 3) FE-Ba 0.091 0.012 *Sarcosine (position 3) FESar 0.160 0.034 * γAbu (position 3) FE(γAbu)0.130 0.050 * Dipeptide FE 0.105 0.014 * Dipeptide: D-phe-D-glu fe 0.1610.010 * Dipeptide: D-cha-D-glu (cha)e 0.189 0.020 * L-Tyr YEG 0.3060.032 NS L-Phe-L-Asp FDG 0.262 0.042 NS D-Phe-D-Asp fdG 0.395 0.105 NSL-Tyr-L-Asp YDG 0.221 0.106 NS D-Tyr-D-Asp ydG 0.208 0.083 NSD-Trp-D-Glu weG 0.201 0.047 NS D-Trp-D-Asp wdG 0.200 0.043 NS Dipeptide:L-Glu-Gly EG 0.242 0.085 NS L-Glu EEG 0.230 0.015 NS Gly GEG 0.202 0.081NS L-Ala AEG 0.252 0.106 NS L-Leu LEG 0.196 0.050 NS L-Ile IEG 0.2220.070 NS L-Val VEG 0.313 0.030 NS L-Nval (Nval)EG 0.191 0.082 NS L-ThrTEG 0.250 0.042 NS L-Cys CEG 0.247 0.107 NS L-His HEG 0.195 0.028 NSL-Asp NEG 0.384 0.122 NS L-Arg REG 0.220 0.047 NS L-Pro PEG 0.324 0.072NS L-Ala FAG 0.171 0.040 NS L-Gln FQG 0.309 0.050 NS L-Asp FDG 0.2620.042 NS D-Tyr-D-Asp ydG 0.208 0.083 NS L-Lys FKG 0.183 0.027 NS L-ArgFRG 0.160 0.025 NS L-His FHG 0.167 0.050 NS L-Hse (position 2) F(Hse)G0.258 0.050 NS Pro (position 3) FEP 0.246 0.021 NS L-Ala (position 3)FEA 0.217 0.046 NS L-Nval FE(Nval) 0.241 0.037 NS L-Gln FEQ 0.310 0.046NS L-Thr FET 0.237 0.054 NS L-Glu FEE 0.318 0.056 NS L-Lys FEK 0.2520.056 NS L-Arg FER 0.134 0.023 NS L-His FEH 0.200 0.035 NS L-Cys FEC0.187 0.023 NS Ac-FEG 0.173 0.046 NS FEG-NH₂ 0.262 0.081 NS

We claim:
 1. A method of inhibiting an inflammatory reaction in a mammalcomprising administering to the mammal an effective amount of a peptideof the formula: X¹—X²—X³ wherein X¹ is selected from the groupconsisting of 2-amino-hexanoic acid; 2-amino-heptanoic acid;2-amino-octanoic acid; cyclohexyl-substituted 2-amino-ethanoic acid,2-amino-propanoic acid or 2-amino-butanoic acid and methionine; X² is anacidic amino acid; and X³ is an aliphatic amino acid; or of a peptide ofthe formula: X⁴ —X⁵ wherein X⁴ is an aromatic or aliphatic amino acid;and X⁵ is an acidic amino acid.
 2. The method of claim 1 wherein theadministered peptide is of the formula X¹—X²—X³ wherein X¹ is selectedfrom the group consisting of 2-amino-hexanoic acid, 2-amino-heptanoicacid, 2-amino-octanoic acid, 2-amino-2-cyclohexyl ethanoic acid,2-amino-3-cyclohexyl propanoic acid, 2-amino-3-cyclohexyl butanoic acid,2-amino-4-cyclohexyl butanoic acid and methionine; X² is glutamic acid;and X³ is selected from the group consisting of glycine, methionine,isoleucine, alanine, β-alanine, sarcosine and γ-aminobutyric acid. 3.The method of claim 2 wherein X¹ is selected from the group consistingof 2-amino-3-cyclohexyl propanoic acid (cyclohexylalanine),2-amino-hexanoic acid (norleucine) and methionine.
 4. The method ofclaim 1 wherein the administered peptide is of the formula X⁴—X⁵ whereinX⁴ is selected from the group consisting of phenylalanine, norleucine,cyclohexylalanine, methionine and tyrosine; and X⁵ is glutamic acid. 5.The method of claim 1 wherein at least one amino acid of the peptide isa D-amino acid.
 6. The method of claim 1 wherein the administeredpeptide is selected from the group consisting of (a)L-cyclohexylalanine-L-glutamic acid-glycine; (b)D-cyclohexylalanine-D-glutamic acid-glycine; (c) L-norleucine-L-glutamicacid-glycine; (d) D-norleucine-D-glutamic acid-glycine; (e)L-methionine-L-glutamic acid-glycine; (f) D-methionine-D-glutamicacid-glycine; (g) L-cyclohexylalanine-L-glutamic acid-L-methionine; (h)D-cyclohexylalanine-D-glutamic acid-L-methionine; (i)L-cyclohexylalanine-L-glutamic acid-L-isoleucine; (j)D-cyclohexylalanine-D-glutamic acid-D-isoleucine; (k)L-cyclohexylalanine-L-glutamic acid; (l) D-cyclohexylalanine-D-glutamicacid; (m) L-norleucine-L-glutamic acid; (n) D-norleucine-L-glutamicacid; (o) L-methionine-L-glutamic acid; (p) D-methionine-D-glutamicacid; (q) L-phenylalanine-L-glutamic acid; (r)D-phenylalanine-D-glutamic acid; and (s) D-tyrosine-D-glutamic acid. 7.The method of claim 1 wherein the administered peptide isL-cyclohexylalanine-L-glutamic acid-glycine.
 8. The method of claim 1wherein the administered peptide is D-cyclohexylalanine-D-glutamicacid-glycine.
 9. The method of claim 1 wherein the inflammatory reactionis associated with a disorder selected from the group consisting ofasthma, rhinitis, conjunctivitis, atopic dermatitis and intestinalanaphylaxis.
 10. The method of claim 1 wherein the inflammatory reactionis a Type I, Type II or Type III hypersensitivity reaction.
 11. Themethod of claim 1 wherein the inflammatory reaction is an immunologicalreaction.
 12. The method of claim 1 wherein the C-terminal amino acid ofthe peptide, X³ or X⁵, is amidated.
 13. The method of claim 1 whereinthe amide of the N-terminal amino acid of the peptide, X¹ or X⁴, islower alkyl-substituted, preferably methylated.
 14. A method of treatingasthma in a mammal comprising administering to the mammal an effectiveamount of a peptide of the formula: X¹—X²—X³ wherein X¹ is selected fromthe group consisting of 2-amino-hexanoic acid; 2-amino-heptanoic acid;2-amino-octanoic acid; cyclohexyl-substituted 2-amino-ethanoic acid,2-amino-propanoic acid or 2-amino-butanoic acid and methionine; X² is anacidic amino acid; and X³ is an aliphatic amino acid; or of a peptide ofthe formula: X⁴ —X⁵ wherein X⁴ is an aromatic or aliphatic amino acid;and X⁵ is an acidic amino acid.
 15. The method of claim 14 wherein theadministered peptide is of the formula X¹—X²—X³ wherein X¹ is selectedfrom the group consisting of 2-amino-hexanoic acid, 2-amino-heptanoicacid, 2-amino-octanoic acid, 2-amino-2-cyclohexyl ethanoic acid,2-amino-3-cyclohexyl propanoic acid, 2-amino-3-cyclohexyl butanoic acid,2-amino-4-cyclohexyl butanoic acid and methionine; X² is glutamic acid;and X³ is selected from the group consisting of glycine, methionine,isoleucine, alanine, P-alanine, sarcosine and y-aminobutyric acid. 16.The method of claim 15 wherein X¹ is selected from the group consistingof 2-amino-3-cyclohexyl propanoic acid (cyclohexylalanine),2-amino-hexanoic acid (norleucine) and methionine.
 17. The method ofclaim 14 wherein the administered peptide is of the formula X⁴ —X⁵wherein X⁴ is selected from the group consisting of phenylalanine,norleucine, cyclohexylalanine, methionine and tyrosine; and X⁵ isglutamic acid.
 18. The method of claim 14 wherein at least one aminoacid of the peptide is a D-amino acid.
 19. The method of claim 14wherein the administered peptide is selected from the group consistingof (a) L-cyclohexylalanine-L-glutamic acid-glycine; (b)D-cyclohexylalanine-D-glutamic acid-glycine; (c) L-norleucine-L-glutamicacid-glycine; (d) D-norleucine-D-glutamic acid-glycine; (e)L-methionine-L-glutamic acid-glycine; (f) D-methionine-D-glutamicacid-glycine; (g) L-cyclohexylalanine-L-glutamic acid-L-methionine; (h)D-cyclohexylalanine-D-glutamic acid-L-methionine; (i)L-cyclohexylalanine-L-glutamic acid-L-isoleucine; (j)D-cyclohexylalanine-D-glutamic acid-D-isoleucine; (k)L-cyclohexylalanine-L-glutamic acid; (l) D-cyclohexylalanine-D-glutamicacid; (m) L-norleucine-L-glutamic acid; (n) D-norleucine-L-glutamicacid; (o) L-methionine-L-glutamic acid; (p) D-methionine-D-glutamicacid; (q) L-phenylalanine-L-glutamic acid; (r)D-phenylalanine-D-glutamic acid; and (s) D-tyrosine-D-glutamic acid. 20.The method of claim 14 wherein the administered peptide isL-cyclohexylalanine-L-glutamic acid-glycine.
 21. The method of claim 14wherein the administered peptide is D-cyclohexylalanine-D-glutamicacid-glycine.
 22. The method of claim 14 wherein the C-terminal aminoacid of the peptide, X³ or X⁵, is amidated.
 23. The method of claim 14wherein the amide of the N-terminal amino acid of the peptide, X¹ or X⁴,is lower alkyl-substituted, preferably methylated.
 24. The method ofclaim 1 wherein the mammal is a human.
 25. A peptide of the formula:X¹—X²—X³ wherein X¹—X²—X³ wherein X¹ is selected from the groupconsisting of 2-amino-hexanoic acid; 2-amino-heptanoic acid;2-amino-octanoic acid; cyclohexyl-substituted 2-amino-ethanoic acid,2-amino-propanoic acid or 2-amino-butanoic acid and methionine; X² is anacidic amino acid; and X³ is an aliphatic amino acid.
 26. The peptide ofclaim 25 wherein the peptide is of the formula X¹—X²—X³ wherein X¹ isselected from the group consisting of 2-amino-hexanoic acid,2-amino-heptanoic acid, 2-amino-octanoic acid, 2-amino-2-cyclohexylethanoic acid, 2-amino-3-cyclohexyl propanoic acid, 2-amino-3-cyclohexylbutanoic acid, 2-amino-4-cyclohexyl butanoic acid and methionine; X² isglutamic acid; and X³ is selected from the group consisting of glycine,methionine, isoleucine, alanine, β-alanine, sarcosine and γ-aminobutyricacid.
 27. The peptide of claim 25 wherein the peptide is selected fromthe group consisting of (a) L-cyclohexylalanine-L-glutamic acid-glycine;(b) D-cyclohexylalanine-D-glutamic acid-glycine; (c)L-norleucine-L-glutamic acid-glycine; (d) D-norleucine-D-glutamicacid-glycine; (e) L-methionine-L-glutamic acid-glycine; (f)D-methionine-D-glutamic acid-glycine; (g) L-cyclohexylalanine-L-glutamicacid-L-methionine; (h) D-cyclohexylalanine-D-glutamic acid-L-methionine;(i) L-cyclohexylalanine-L-glutamic acid-L-isoleucine; (j)D-cyclohexylalanine-D-glutamic acid-D-isoleucine.
 28. A peptide of theformula: X⁴—X⁵ wherein X⁴ is an aromatic or aliphatic amino acid; and X⁵is an acidic amino acid.
 29. The peptide of claim 27 wherein the peptideis selected from the group consisting of (a)L-cyclohexylalanine-L-glutamic acid; (b) D-cyclohexylalanine-D-glutamicacid; (c) L-norleucine-L-glutamic acid; (d) D-norleucine-L-glutamicacid; (e) L-methionine-L-glutamic acid; (f) D-methionine-D-glutamicacid; and (g) D-tyrosine-D-glutamic acid.
 30. The peptide of claim 25wherein at least one amino acid is a D amino acid.
 31. The peptide ofclaim 28 wherein at least one amino acid is a D amino acid.
 32. Thepeptide of claim 25 wherein the C-terminal amino acid of the peptide, X³or X⁵, is amidated.
 33. The peptide of claim 25 wherein the amide of theN-terminal amino acid of the peptide, X¹ or X⁴, is loweralkyl-substituted, preferably methylated.
 34. A pharmaceuticalcomposition comprising a peptide of claim 25 and a pharmaceuticallyacceptable carrier.
 35. A pharmaceutical composition comprising apeptide of claim 28 and a pharmaceutically acceptable carrier.
 36. Apharmaceutical composition comprising a peptide of claim 27 and apharmaceutically acceptable carrier.
 37. A pharmaceutical compositioncomprising a peptide of claim 29 and a pharmaceutically acceptablecarrier.